4,4'-methylenedianiline

Carcinogenicity

Description of key information

The key studies for carcinogenicity were performed similar to OECD 451 (NTP, 1983; Weisburger et al. 1984; Lamb et al. 1986). Rats and mice were treated with 4,4‘-MDA for 103 weeks and the liver and thyroid were the target organs for neoplastic and non-neoplastic findings. Clear evidence for the carcinogenic potential of the substance in both species was provided by the study data. In rats significantly increased incidences of thyroid follicular cell carcinomas in males, thyroid follicular cell adenoma in females (both exceeding the historical control data range) and neoplastic nodules in the liver of males were seen. Cystic and hyperplastic follicular thyroid lesions were increased in females. In mice, significantly increased incidences of thyroid follicular cells adenoma (males and females) as well as liver cell carcinoma (males) were observed, which exceeded the historical control data range. The incidences of liver cell adenoma and carcinoma were also significantly increased in females; however the incidences were still within the historical control data range. The neoplastic lesions were accompanied by liver degeneration in both sexes.

The outcome of the key studies was supported by rat studies applying an initiation-promotion protocol in rats. Repeated dosing with 4,4‘-MDA after an initiating event revealed a promoting effect on the tumor development in liver and thyroid as shown by increased incidences of non-neoplastic and neoplastic lesions in the target organs (Uwagawaet al., 1992; Fukushima et al., 1991; Hiasa et al., 1984). 

Data on the carcinogenic potential of 4,4’-MDA in humans indicate single cases of bladder cancer in workers exposed to the substance (Liss et al., 1994; NIOSH, 1982).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug. 1978 - Sep. 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

- only 2 doses tested

Principles of method if other than guideline:

The study design conformed to the NCI guidelines for carcinogen bioassays in small rodents (Sontag et al. (1976), Carcinogenesis Technical Report Series 1, DHEW publication 76-801), and was similar to OECD Guideline 451.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Kodak Company, Rochester; Lot No. A8
- Purity: 98.6 %

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The mice were 9 weeks old as received, and were observed for 19 days before the start of the study. The temperature was 20-28°C, the uncontrolled humidity was 8-78 % RH (average 41 % RH), and there were 10-12 air changes/hr. The average water consumption per animal, was calculated by dividing the total water consumption measured for all cages by the number of surviving animals in the group. The drinking water for the control animals was adjusted to the same pH as that of the 300 ppm formulation, which averaged pH 3.7.

Route of administration:

oral: drinking water

Vehicle:

other: drinking water adjusted with 0.1 N HCl to pH 3.7 of the 300 ppm formulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of formulated drinking water gave means of 152.1 and 290.3 ppm MDA, respectively. The nominal concentration 150 ppm corresponds to the following average calculated amounts of MDA, consumed via the drinking water: 25 mg/kg bw/day for males and 19 mg/kg bw/day for females.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

continuous

Post exposure period:

1-2 weeks

Dose / conc.:

150 ppm (nominal)

Remarks:

dosage expressed as the free base

Dose / conc.:

300 ppm (nominal)

Remarks:

dosage expressed as the free base

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

All animals were observed twice daily for signs of morbidity and mortality. Clinical signs were recorded monthly. Body weights by cage were recordedevery week for the first 13 weeks and monthly thereafter.

Sacrifice and pathology:

Moribund animals and animals that survived to the end of the study were killed and necropsied. Necropsies were also performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. Histopathological examination was done on 38+ tissues from each mouse.

Statistics:

Tumor incidence statistically analyzed by methods of Peto et al. (1980), Fisher exact test for pairwise comparisons and Cochran-Armitage linear trend test for dose-response trends.

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY:
No compound-related clinical effects were observed. There was reduced survival amongst the males receiving the higher dose of MDA (control: 40/50; high dose: 32/50).

BODY WEIGHT AND WEIGHT GAIN:
The high dose groups had reduced mean body weights throughout most of the study; this difference was first noticeable by week 16.

WATER CONSUMPTION AND COMPOUND INTAKE :
Compared with the controls the average daily water consumption of the male mice was 106 % (150 ppm MDA) and 111 % (300 ppm MDA).  The corresponding figures for females were 87 %, and 96 %, respectively. The average calculated amounts of MDA, consumed via the drinking water by males, were 25 (150 ppm MDA) and 57 (300 ppm MDA) mg/kg bw/day.  For the female mice, the corresponding figures were 19 and 43 mg/kg bw/day, respectively.

HISTOPATHOLOGY - NON-NEOPLASTIC AND NEOPLASTIC:
Both neoplastic and non-neoplastic findings were observed in the thyroid gland of mice receiving MDA (for incidences see table 1 in "remarks on results including tables and figures). The incidences of thyroid neoplasms in the high dose groups were elevated compared with those of control groups of both sexes. Follicular cell adenoma was increased in high dose male and female mice as compared with controls. In females 2/50 follicular cell carcinomas were observed. The non-neoplastic effect in the thyroid was follicular cell hyperplasia in high dose male and female mice. In addition, neoplastic and non-neoplastic effects were observed in the liver of mice receiving MDA as indicated in table 1 (see "remarks on results including tables and figures). Hepatocellular adenoma was increased in a dose-related manner in dosed females. Hepatocellular carcinoma was observed in greater incidence in males at 150 and 300 ppm and in high dose females. Liver degeneration was present in 80 % of the low dose and 60 % of the high dose males as well as in 14 % of the high dose females, but was not found in control groups.

Other tumors that were elevated in dosed mice included adrenal pheochromocytomas in males (2/48, 12/49, 14/49), alveolar/bronchiolar adenoma in females (1/50, 2/50, 6/49) and malignant lymphomas in females (13/50, 28/50, 29/50). Decreases in tumor incidences were observed for alveolar or bronchiolar adenomas (combined) in male mice (12/49, 9/49, 3/49).

Nephropathy was increased in dosed mice of both sexes (males: 18/49, 34/50, 36/50; females: 6/50, 21/50, 35/50); renal papillary mineralization was greater in males (12/50) and females (14/50) at 300 ppm compared to controls.

Key result

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Key result

Dose descriptor:

LOAEL

Effect level:

150 ppm (nominal)

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Number of mice with non-neoplastic and neoplastic lesions in the thyroid and liver

Sex		Males			Females
Dose in ppm	0	150	300	0	150	300
T h y r o i d :
Follicular cell hyperplasia	0/47	3/49	18/49	0/50	0/47	23/50
Follicular cell adenoma	0/47	3/49	16/49*	0/50	1/47	13/50*
Follicular cell carcinoma	0/47	0/49	0/49	0/50	0/47	2/50
L i v e r :
Liver cell degeneration	0/50	40/50	30/50	0/50	0/50	7/50
Liver cell adenoma				3/50	9/50	12/50*
Liver cell carcinoma	10/49	33/50*	29/50*	1/50	6/50	11/50*

* Tumor incidences indicated as statistically significant; no statistics available on the non-neoplastic lesions

                                                                                                             

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug. 1978 - Sep. 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

- only 2 doses tested

Principles of method if other than guideline:

The study design conformed to the NCI guidelines for carcinogen bioassays in small rodents (Sontag et al., (1976), Carcinogenesis Technical Report Series 1, DHEW publication 76-801), and was similar to OECD Guideline 451.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Kodak Company, Rochester; Lot No. A8
- Purity: 98.6 %

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were 4 weeks old as received, and were observed for 2 weeks before the start of the study. The temperature was 20-28°C, the uncontrolled humidity was 8-78 % RH (average 41 % RH), and there were 10-12 air changes/hr. The average water consumption per animal was calculated by dividing the total water consumption measured for all cages by the number of surviving animals in the group. The drinking water for the control animals was adjusted to the same pH as that of the 300 ppm formulation, which averaged pH 3.7.

Route of administration:

oral: drinking water

Vehicle:

other: drinking water adjusted with 0.1 N HCl to pH 3.7 of the 300 ppm formulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of formulated drinking water gave means of 152.1 and 290.3 ppm MDA, respectively. The nominal concentration 150 ppm corresponds to the following average calculated amounts of MDA, consumed via the drinking water: 9 mg/kg bw/day for males and 10 mg/kg bw/day for females.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

continuous

Post exposure period:

2-3 weeks

Dose / conc.:

150 ppm (nominal)

Remarks:

dosage expressed as the free base

Dose / conc.:

300 ppm (nominal)

Remarks:

dosage expressed as the free base

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

All animals were observed twice daily for signs of morbidity and mortality. Clinical signs were recorded monthly. Body weights by cage were recorded every week for the first 13 weeks and monthly thereafter.

Sacrifice and pathology:

Moribund animals and animals that survived to the end of the study were killed and necropsied. Necropsies were also performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. Histopathological examination was done on 37+ tissues from each rat.

Statistics:

Tumor incidence statistically analyzed by methods of Peto et al. (1980), Fisher exact test for pairwise comparisons and Cochran-Armitage linear trend test for dose-response trends.

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY:
No compound-related clinical effects were observed. No significant differences in survival were seen between any groups of either sex.

BODY WEIGHT AND WEIGHT GAIN:
The female rats receiving water containing 300 ppm MDA had lower final mean body weights than the controls. The other groups had normal body weights.

WATER CONSUMPTION AND COMPOUND INTAKE :
Compared with the controls the average daily water consumption of the male rats was 87 % (150 ppm MDA) and 75 % (300 ppm MDA).  The corresponding figures for females were 93 %, and 82 %, respectively. The average calculated amounts of MDA, consumed via the drinking water by males, were 9 (150 ppm MDA) and 16 (300 ppm MDA) mg/kg bw/day.  For the female rats, the corresponding figures were 10 and 19 mg/kg bw/day, respectively.

HISTOPATHOLOGY - NON-NEOPLASTIC AND NEOPLASTIC:
Both neoplastic and non-neoplastic findings were observed in the thyroid gland of rats receiving MDA (for incidences see table 1 in "remarks on results including tables and figures). Both follicular cell and C-cell neoplasms were found. Follicular cell carcinomas were increased in males and follicular cell adenomas were dose-related increased in females. C-cell adenomas occurred with a positive trend in females. The non-neoplastic effects in the thyroid were follicular cysts and follicular cell hyperplasia in both sexes. In addition, neoplastic and non-neoplastic effects were observed in the liver of rats receiving MDA as indicated in table 1 (see "remarks on results including tables and figures"). Neoplastic nodules of the liver were statistically significantly increased in low and high dose males and there was an slightly increased incidence in females at 150 and 300 ppm. Non-neoplastic liver lesions included unspecified dilatation, fatty metamorphosis, and focal cellular change.

Males had a dose-related increase in kidney mineralization (control: 9/50; low dose: 10/50; high dose: 19/50) . Furthermore, there was a negative trend effect on leukaemia in male rats. Uncommon tumors were observed in dosed animals at low incidences: bile duct adenoma in 1/50 high dose males, transitional cell papillomas of the urinary bladder in females (low dose: 2/50; high dose: 1/50) and granulosa cell tumors of the ovary in females (low dose: 3/50; high dose: 2/50).

Key result

Dose descriptor:

NOAEL

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Key result

Dose descriptor:

LOAEL

Effect level:

150 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Number of rats with non-neoplastic and neoplastic lesions in the thyroid and liver

Sex		Males			Females
Dose in ppm	0	150	300	0	150	300
T h y r o i d :
Follicular cysts	1/49	2/47	3/48	0/47	3/47	7/48
Follicular cell hyperplasia	1/49	2/47	3/48	1/47	3/47	8/48
Follicular cell adenoma	1/49	4/47	3/48	0/47	2/47	17/48*
Follicular cell carcinoma	0/49	0/47	7/48*	0/47	2/47	2/48
C-Cell adenoma	0/49	0/47	0/48	0/47	3/47	6/48
L i v e r :
Unspecified dilatation	1/50	6/50	10/50
Fatty metamorphosis	14/50	28/50	33/50	7/50	20/50	11/50
Focal cellular change	14/50	38/50	36/50	5/50	17/50	10/50
Neoplastic nodules	1/50	12/50*	25/50*	4/50	8/50	8/50

* Tumor incidences indicated as statistically significant; no statistics available on the non-neoplastic lesions

                                                                                                             

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

9 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

similar to OECD TG 451

System:

hepatobiliary

Organ:

liver

thyroid gland

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A causal relationship has been established between 4,4’-MDA and an increased incidence of benign and malignant neoplasms in both sexes of the rat and mouse. The single cases of bladder cancer reported in humans do not represent sufficient evidence of carcinogenicity in humans. Overall, the available data represent sufficient evidence to demonstrate animal carcinogenicity (presumed human carcinogen) resulting in the classification and labeling of 4,4’-MDA as carcinogenic, cat 1B (H350: may cause cancer) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Additional information

Oral Administration

The key study for carcinogenicity was performed similar to OECD TG 451 (two dose groups) in F344 rats and B6C3F1 mice (NTP, 1983; Weisburger et al., 1984, Lamb et al., 1986). Both species were administered with doses of 0, 150 or 300 ppm test substance via the drinking water for 103 weeks. The calculated average test substance intake for the rats was 9 or 16 mg/kg bw/d for males and 10 or 19 mg/kg bw/d for females (low and high dose, respectively). For the mice, doses of 25 or 57 mg/kg bw/d (males) and 19 or 43 mg/kg bw/d (females), were calculated.

In rats, no treatment related mortality or clinical signs were observed. Survival was comparable among all groups. The high dose female rats had lower final mean body weights than the controls. The other groups had normal body weights. Histopathological examination of the rats revealed significantly increased incidences of thyroid follicular cell carcinomas in males, thyroid follicular cell adenomas in females and not statistically significant C-cell adenomas of the thyroid gland in females in the high dose group. Cystic and hyperplastic follicular thyroid lesions were increased in high dose females. At the low and high dose group neoplastic nodules in the liver of males were observed. In addition, rats of each dose group showed toxic liver effects (fatty metamorphosis, focal cellular change). An increased incidence of renal mineralization was evident in high dose males.

 

Historical control data from the NTP Historical Controls Database were also considered and are presented in Table 2. They consist of chronic toxicity studies with substance administration via drinking water which were performed between 1984 and 1994. Specific data covering an earlier time period are not available in the database. The incidence of thyroid follicular cell carcinoma in male rats (7/48) and adenoma in female rats (17/48) were above the historical control data range (0/51 - 2/50 and 0/80 - 3/50, respectively). For the observed C-cell adenomas of the thyroid gland in females in the 300 ppm dose group (6/48) the cases were in the historical control data range (2/50 -13/80).

 

Table 1: Number of rats with non-neoplastic and neoplastic lesions in the thyroid and liver

	Male			Female
Dose (ppm)	0	150	300	0	150	300
Thyroid
Follicular cysts	1/49	2/47	3/48	0/47	3/47	7/48
Follicular cell hyperplasia	1/49	2/47	3/48	1/47	3/47	8/48
Follicular cell adenoma	1/49	4/47	3/48	0/47	2/47	17/48*
Follicular cell carcinoma	0/49	0/47	7/48*	0/47	2/47	2/48
C-Cell adenoma	0/49	0/47	0/48	0/47	3/47	6/48
Liver
Focal cellular change	14/50	38/50	36/50	5/50	17/50	10/50
Neoplastic nodules	1/50	12/50*	25/50*	4/50	8/50	8/50
* Tumor incidences indicated as statistically significant; no statistics available on the non-neoplastic lesions

 

 

Table 2: NTP Historical Control Data (tumor incidence summary, Fischer 344 rat, 1984 - 1999)

THYROID GLAND: Follicular cell                                                           Fischer 344 rats
(Updated 12/99)		Oral water Males (6 studies)	Females (6 studies)
Adenoma	Total	4/330 (1.2%)	5/329 (1.5%)
	Mean (sd)	1.3% (1.6)	1.7% (2.3)
	Range	0/51 – 2/50	0/80 – 3/50
Males 0/51  1/49  0/80  2/50  1/50  0/50 Females 3/50  1/49  0/80  1/50  0/50  0/50
Carcinoma	Total	3/330 (0.9%)	3/329 (0.9%)
	Mean (sd)	0.9% (1.6)	0.8% (1.2)
	Range	0/51 – 2/50	0/50 – 2/80
Males 0/51  0/49  1/80  2/50  0/50  0/50 Females 0/50  0/49  2/80  1/50  0/50  0/50
Carcinoma or Adenoma	Total	7/330 (2.1%)	8/329 (2.4%)
	Mean (sd)	2.2% (3.0)	2.4% (2.3)
	Range	0/51 – 4/50	0/50 – 3/50
Males 0/51  1/49  1/80  4/50  1/50  0/50 Females 3/50  1/49  2/80  2/50  0/50  0/50

THYROID GLAND: C-Cell                                                                     Fischer 344 rats
(Updated 12/99)		Oral water Males (6 studies)	Females (6 studies)
Adenoma	Total	36/330 (10.9%)	34/329 (10.3%)
	Mean (sd)	10.3% (4.5)	9.8% (4.7)
	Range	2/50 – 14/80	2/50 – 13/80
Males 6/51  5/49  14/80  4/50  5/50  2/50 Females 4/50  5/49  13/80  7/50  2/50  3/50
Carcinoma	Total	3/330 (0.9%)	4/329 (1.2%)
	Mean (sd)	0.9% (1.0)	1.2% (1.0)
	Range	0/49 – 1/51	0/50 – 1/50
Males 1/51  0/49  1/80  1/50  0/50  0/50 Females 1/50  1/49  1/80  1/50  0/50  0/50
Carcinoma or Adenoma	Total	39/330 (11.8%)	37/329 (11.2%)
	Mean (sd)	11.1% (4.9)	10.6% (5.3)
	Range	2/50 – 15/80	2/50 – 14/80
Males 7/51  5/49  15/80  5/50  5/50  2/50 Females 5/50  5/49  14/80  8/50  2/50  3/50

In mice, no compound-related clinical effects were observed. There was reduced survival amongst the high-dose males. Commencing at week 16, the high-dose groups had reduced mean body weights throughout most of the study.

Both neoplastic and non-neoplastic findings were observed in the thyroid gland of treated mice. Follicular cell adenoma was statistically significantly increased in high dose male and female mice as compared with controls. In 2/50 females follicular cell carcinomas were observed. The non-neoplastic effect in the thyroid was follicular cell hyperplasia in high dose male and female mice. In addition, neoplastic and non-neoplastic effects were observed in the liver of treated mice. Hepatocellular adenoma was increased in a dose-related manner in dosed females reaching statistical significance at both dose levels. Hepatocellular carcinoma was observed in greater incidence and with statistical significance in males of both dose groups and in high dose females. Liver degeneration was present in 80% of the low dose and 60% of the high dose males as well as in 14% of the high dose females, but was not found in control groups. Other tumors that were elevated in dosed mice included adrenal pheochromocytomas in males (2/48, 12/49, 14/49), alveolar/bronchiolar adenoma in females (1/50, 2/50, 6/49) and malignant lymphomas in females (13/50, 28/50, 29/50). Decreases in tumor incidences were observed for alveolar or bronchiolar adenomas (combined) in male mice (12/49, 9/49, 3/49). Nephropathy was increased in dosed mice of both sexes (males: 18/49, 34/50, 36/50; females: 6/50, 21/50, 35/50); renal papillary mineralization was greater in males (12/50) and females (14/50) at the high dose group when compared to controls.

Historical control data from the NTP Historical Controls Database were also considered and are presented in Table 4. They consist of chronic toxicity studies with substance administration via the drinking water which were performed between 1984 and 1994. Specific data covering an earlier time period are not available in the database. The number of observed thyroid follicular cell adenoma in male (16/49) and female (13/50) mice at the highest dose level were above the historical control data ranges (males: 1/49-2/49 and females: 0/50-3/50). Furthermore, the incidence of liver cell carcinoma in males at 150 ppm (33/50) and 300 ppm (29/50) exceeded the historical control ranges (5/51 to 25/59). For females the significant increase in liver cell carcinoma at the highest dose level (11/50) was within the historical control data range (4/50-25/59) as were the significantly increased numbers of liver cell adenoma at 150 ppm (9/50) and 300 ppm (11/50) (historical control data range 13/50 – 40/50).

Table 3: Number of mice with non-neoplastic and neoplastic lesions in the thyroid and liver

	Male			Female
Dose (ppm)	0	150	300	0	150	300
Thyroid
Follicular cell hyperplasia	0/47	3/49	18/49	0/50	0/47	23/50
Follicular cell adenoma	0/47	3/49	16/49*	0/50	1/47	13/50*
Follicular cell carcinoma	0/47	0/49	0/49	0/50	0/47	2/50
Liver
Liver cell degeneration	0/50	40/50	30/50	0/50	0/50	7/50
Liver cell adenoma	7/49	10/50	8/50	3/50	9/50*	12/50*
Liver cell carcinoma	10/49	33/50*	29/50*	1/50	6/50	11/50*
* Tumor incidences indicated as statistically significant; no statistics available on the non-neoplastic lesions

 

 

Table 4: NTP Historical Control Data (tumor incidence summary, B6C3F1 mice, 1984 - 1999)

THYROID GLAND: Follicular cell                                                                B6C3F1 mice
(Updated 12/99)		Oral water Males (6 studies)	Females (6 studies)
Adenoma	Total	7/339 (2.1%)	12/338 (3.6%)
	Mean (sd)	2.2% (1.0)	3.4% (2.2)
	Range	1/79 – 2/49	0/50 – 3/50
Males 1/50  1/51  1/79  1/60  1/50  2/49 Females 2/50  0/50  4/80  2/58  1/50  3/50
Carcinoma	Total	0/339 (0%)	0/338 (0%)
	Mean (sd)	0.0% (0.0)	0.0% (0.0)
	Range	0/50 – 0/50	0/50 – 0/50
Males 0/50  0/51  0/79  0/60  0/50  0/49 Females 0/50  0/50  0/80  0/58  0/50  0/50
Carcinoma or Adenoma	Total	7/339 (2.1%)	12/338 (3.6%)
	Mean (sd)	2.2% (1.0)	3.4% (2.2)
	Range	0/79 – 2/49	0/50 – 3/50
Males 1/50  1/51  1/79  1/60  1/50  2/49 Females 2/50  0/50  4/80  2/58  1/50  3/50

LIVER                                                                                                        B6C3F1 mice
(Updated 12/99)		Oral water Males (6 studies)	Females (6 studies)
Hepatocellular Adenoma	Total	208/339 (61.4%)	187/338 (55.3%)
	Mean (sd)	61.0% (8.2)	55.0% (21.2)
	Range	24/51 – 35/50	13/50 – 40/50
Males 30/50  24/51  50/79  40/59  35/50  29/50 Females 19/50  13/50  49/80  29/59  40/50  37/49
Hepatocellular Carcinoma	Total	95/339 (28%)	68/338 (20.1%)
	Mean (sd)	27.3% (10.7)	19.7% (12.8)
	Range	5/51 – 25/59	4/50 – 25/59
Males 12/50  5/51  25/79  25/59  13/50  15/50 Females  6/50  4/50  14/80  25/59  6/50  13/49

It can be concluded that, under the conditions of this study, 4,4’-MDA was carcinogenic in rats and mice, inducing thyroid and liver tumors. The LOAEL in both species was 150 ppm. (National Toxicology Program, NTP-81-143, NIH Publ. No. 83-2504; NTP TR 248, 1983)

The outcome of the key study is supported by the results of two studies using a wide spectrum organ carcinogenesis model and applying an initiation – promotion protocol (Uwagawa et al., 1992; Fukushima et al., 1991).

 

In a subchronic feeding study in young adult male F344 rats the animals were randomly divided into 3 groups and the following initiation – promotion protocol was applied for testing:

Group 1: sequential treatment with N-Bis(2-hydroxypropyl)nitrosamine (DHPN, 1000 mg/kg bw, i.p., twice on days 1 and 4 of week 1), N-ethyl-N-hydroxyethylnitrosamine (EHEN, 1500 mg/kg bw, i.g., twice on days 8 and 11 of week 2) and 3,2'-dimethyl-4-aminobiphenyl (DMAB, 75 mg/kg bw, s.c., twice on days 15 and 18 of week 3) for initiation (DED regimen). Thereafter, 4,4’-MDA was administered via food at a concentration of 0.1% (promotion) for 12 weeks.

Group 2: sequential treatment with the above mentioned Group 1 substances (DED regimen), then given basal diet until termination.

Group 3: treatment with solvent vehicles of the DED regimen, and then given the test substance as in Group 1.

No mortality was observed. The final body weights of rats given the test substance were significantly lower than in the control. Food and water consumption were normal. Immunohistochemically demonstrated GST-P-positive liver cell foci were assessed as preneoplastic end-point lesions. The test substance significantly enhanced the number and area of GST-P-positive foci per cm² as compared to the DED regimen alone values (Group 2) as well as the test chemical alone values (Group 3). The incidences of thyroid follicular hyperplasia in test substance treated animals (100%, 15/15 animals) with the DED regimen (Group 1) were significantly higher than that in Group 2 treated with DED alone (0%, 0/15 rats). In the treatment group, the incidences of follicular adenomas (100%, 15/15 rats) as well as follicular adenocarcinomas (100%, 15/15 rats) were higher than those of Group 2 treated with DED alone or Group 3 treated with the test substance alone. All other investigated organs did not show signs of neoplastic lesions.

The study confirmed clear modifying influences for the test substance on carcinogenesis in their target organs after sequential pretreatment of animals with different initiating substances. Enhancing effects of 4,4-MDA on preneoplastic and neoplastic lesions were seen in the liver and thyroid. (Uwagawa et al., Jpn. J. Cancer Res. 83, 812-820, 1992)

The modifying potential of various chemicals, including 4,4’-MDA on tumor development was investigated in a wide-spectrum organ carcinogenesis model using male F344/DuCrj rats (initiation-promotion protocol). The animals were treated according to the following schedule:

Group 1: sequential treatment with N-nitrosodiethylamine (DEN; 100 mg/kg body weight, ip, single injection), N-methyl-N-nitrosourea (MNU; 20 mg/kg bw, ip, 4 times during weeks 1 and 2) and N-bis(2-hydroxypropyl)nitrosamine (DHPN; 0.1% in drinking water, during weeks 3 and 4) for initiation (DMD regimen). Thereafter, 4,4-MDA was administered via food at a concentration of 1000 ppm (promotion) for 16 weeks.

Group 2: sequential treatment with the above mentioned Group 1 substances (DMD regimen), then given basal diet until termination.

Group 3: treatment with solvent vehicles of the DMD regimen, and then given the test substance as in Group 1.

All rats were killed at the end of week 20, and the major organs were carefully examined for preneoplastic and neoplastic lesions. Immunohistochemical demonstration of glutathione S--transferase-positive foci was also used for quantitative assessment of liver preneoplastic lesion development. The test substance clearly enhanced the development of GST-P positive foci and the occurrence of thyroid follicular cell hyperplasia, adenoma and carcinoma (present in all animals) after pretreatment with the three carcinogens. (Fukushima et al., Jpn. J. Cancer Res. ,82: 642-649, June 1991)

In another study, 4,4'-MDA promoted the development of thyroid tumors in rats treated with a subeffective dose of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for thyroid tumorigenesis. Male inbred Wistar rats were given a single ip injection of 2800 mg/kg bw of DHPN and fed diets with or without 1000 ppm 4,4’-MDA. Thyroid tumor incidences at the end of week 20 of the experiment were 90% (19/21) in rats given DHPN and then 4,4’-MDA and 28% (6/21) in rats given DHPN alone. The incidence of thyroid cancers was 9.5% (2/21) in rats first given DHPN and then 4,4’-MDA. Untreated rats and rats given 4,4’-MDA alone had no thyroid tumors after 20 weeks. Incidences of kidney tumors were 38% (8/21) in rats given DHPN and then 4,4’-MDA and 28% (6/21) in rats given DHPN alone. No tumors were found in the kidneys and lungs of rats given 4,4’-MDA alone and in those of control rats. Treatment with 4,4’-MDA alone slightly but not significantly decreased the serum concentrations of thyroxine and triiodothyronine; treatment with DHPN plus 4,4’-MDA had no such effect. (Hiasa et al. JNCI, 72 (2): 471-476, 1984)

Other information

The studies summarized below did not follow a validated testing guideline (e.g. OECD TG 451, 453) for chronic toxicity. The documentation of the studies was rather poor and/or the performance of the studies showed significant methodological deficiencies.

In a chronic carcinogenicity study the test substance was applied subcutaneously once per week to Sprague-Dawley rats (male/female) for a period of two years at concentrations of 8.33 or 25 mg/kg bw/d (30 animals per sex per dose). After the exposure period the animals were observed until their death. The test substance did not influence bodyweight development or the life-span of the animals. There was a significant dose-related increase in malignant tumors of the pituitary and thyroid gland in males. (Bayer AG, Report No: 10682, 1981)

In a subchronic carcinogenicity study the test substance was applied subcutaneously every 1 - 3 weeks to Wistar rats (25 males / 25 female) for a period of 705 days at a concentration of 30 - 50 mg/kg bw/injection (total dose: 1.41 g/kg bw). After the exposure period, the animals were observed until their death. The treatment had no effect on the mean life span of the animals. 50% of the treated animals died because of the formation of malignant tumors (control: 26%) in various organs, including liver and thyroid. In total, 33 malign tumors (vehicle control: 16) and 29 benign tumors (vehicle control: 15) were observed. The formation of 4 hepatomas is highlighted.

(Bayer AG, Report No: 1691, 1969 and Steinhoff et al., Naturwissenschaften, 57(5): 247-248, 1970)

In a chronic carcinogenicity study the test substance was applied 3 times/week to the clipped skin of adult C3H mice (male/female) for a period of 24 months at concentrations of 1.12, 2.25 or 4.5 mg/week (40 animals per sex per dose group, 100 animals per sex in control grioup). As positive control substance, Benzo[a]pyrene was applied. There were significant dose-dependent increases in mortality in both sexes. The test substance did not cause any skin tumors. An increase in liver tumors, especially in female mice, was observed. (Oak Ridge National Laboratory, Report No: ORNL/TM-10472, 1987)

In a subchronic to chronic carcinogenicity study the test substance was administered via oral gavage 5 days/week to male rats for a period of 17 weeks at a total dose of 3300 mg/kg bw (24 rats) or 18 months at a total dose of 6000 mg/kg bw (animal number not specified). A control group was not included. After the exposure period, the animals were observed until their death. After the 17-week treatment all animals developed liver cirrhosis. First tumor formation was observed after 2 years (2 benign hepatomas and unspecified tumors in various other tissues). After 18-month treatment, the animals developed 2 tumors in the liver, 1 in the intestines and 1 in the pituitary as well as 2 subcutaneous fibromas. The authors concluded that “neither of the experiments indicated a carcinogenic hazard to workmen handling this compound”.

(Munn et al., Bladder Cancer: a symposium, Aesculapius, Ch.16, 187-93, 1967)

In a subchronic carcinogenicity study the test substance was administered via oral gavage to young adult rats (8 males / 8 females) for a period of 0.3 months (2 doses) or 7.5 months (5 doses) at a dose of 20 mg/animal. After the exposure period the animals were observed for 5 days to 23.5 months. In male rats, receiving 5 doses of the test substance, pronounced liver and kidney lesions were observed. One male rat had a hepatoma with many cells in mitosis, and a kidney tumor of haemangioma-like appearance after 18 months. A female rat had an adenocarcinoma of the uterus after 24 months. Almost all the animals had liver fibrosis and liver inflammation of various grades of severity. (Schoental et al., Israel J. Med. Sci. 4 (6): 1146-58, 1968)

In a subchronic carcinogenicity study the test substance was administered via oral gavage to 20 young adult female Sprague-Dawley rats for a period of 30 days and thereafter observed for 9 months. The test substance was administered in 10 equal doses (every 3 days) at the level of the maximum tolerated dose (300 mg/animal). The control group received the vehicle sesame oil. Histopathological samples of various tissues were prepared. Mammary hyperplasia was observed in 1/14 rats. No mammary adenoma or carcinoma were evident in this study.(Griswold et al., Cancer Res. 28: 924-33, 1968)

In a chronic carcinogenicity study the test substance was administered 3 times/week orally in gel capsules to young adult female beagle dogs for a period of 4 – 7 years in a concentration of 70 mg/d (ca. 4 to 6.26 g/kg bw). The test substance was administered purified or in a crude form (50% test substance + higher molecular weight analogs, 4-5 animals per sex per dose). Moribund dogs, and those that survived until the end of the experiment (7.2 years), were sacrificed, and subjected to post-mortem examination. Cytoscopic examinations were started after 2 years and continued at 15 month intervals. Purified and crude test substance produced similar effects. Moderate to severe gross and histopathological changes in the liver were observed. The lesions ranged from enlarged liver cells and slight structural changes to degeneration, portal fibrosis, liver cell necrosis, haemosiderosis, cell infiltration of the portal areas, dilated bile ducts and thickened bile. Less severe effects were seen in the kidneys and spleen, and occasionally in other organs. Tumors of the urinary bladder or liver did not occur. No relevant effects on hematology and clinical chemistry parameter were observed. (Deichmann et al., Toxicology, 11, 185-188, 1978)

The effects of 4,4’-MDA on the developmental sequence of N-nitrosomorpholine (NNM) induced changes in the rat liver was investigated using a histological, histochemical and morphometric approach. Male Sprague-Dawley rats were treated with NNM for 3 weeks, maintained on basal diet for 1 week and then fed on diets containing 0.08% 4,4’-MDA or, as carcinogen controls, no addition (basal diet) for a further 48 weeks. Control and experimental groups were sacrificed at weeks 4, 16, 28, 40 and 52 of the investigation. 4,4’-MDA did not show clear effects on the development of liver cell carcinoma but enhanced the induction of cholangiofibromas, cholanglofibrosis and, very markedly, spongiosis hepatis. No neoplastic lesions were observed in animals treated with 4,4’-MDA without prior application of NNM. 4,4’-MDA did not significantly influence the number of focal lesions but seemed to effect a decrease in phenotypic alteration within foci. (Ito et al., Carcinogenesis, 5 (3): 335-342, 1984).

 

Information on the inhibitory effects of tumor formation after treatment with 4,4’-MDA is available by Fukushima et al. (1981) and Masui et al. (1986).

 

The effects of 4,4’-MDA administration in the 'post-initiation' stage of liver, kidney and bladder carcinogenesis were examined in male F344 rats. In experiment I, rats were given drinking water containing 0.1% N-ethyl-N-hydroxyethylnitrosamine for 2 weeks then diet containing 0.1% 4,4’-MDA for 32 weeks. In week 3, the right kidney was removed. The incidence of hepatocellular carcinoma was significantly less in rats given 4,4’-MDA than in controls and a decrease in the incidence and average number/cm² of neoplastic nodules and renal cell tumors of the kidney was observed. In experiment II, rats were given 0.01% N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks and then 0.1% 4,4’-MDA for 34 weeks in their drinking water. 4,4’-MDA inhibited the induction of papillomas in the bladder. These results indicate that test substance administration in the 'post-initiation' stage inhibited liver, kidney and bladder carcinogenesis in rats.(Fukushima et al., Carcinogenesis, 2 (10): 1033-1037, 1981)

4,4’-MDA was investigated for its inhibitory effect on hepatocarcinogenesis in male F344 rats. Rats were initially given a single ip injection of diethylnitrosamine (200 mg/kg bw) and fed basal diet containing 0.02% 2-acetylaminofluorene from week 2 to 8. Animals were subjected to partial hepatectomy at the end of week 3. From week 12 to 36 animals were given basal diet containing 0.1% 4,4’-MDA, then killed at week 40, 4 weeks after cessation of treatment. The incidence of hepatocellular carcinoma (HCC) was significantly decreased in the group given 4,4’-MDA. Quantitative analysis of the number and area of HCC per unit liver area revealed a significant decrease in the area of HCC in this group, suggesting an inhibitory effect on the development of HCC. (Masui et al., Jpn. J. Cancer Res. (Gamm), 77: 231-237, 1986)

Toxicogenomic analysis data revealed that this method is not applicable regarding the prediction of tumor formation for 4,4’-MDA (Ellinger-Ziegelbauer et al., 2008).

In a subacute carcinogenicity study the test substance was administered daily via oral gavage to adult male Wistar rats for a period of up to 14 days in a concentration of 50 mg/kg bw/d. RNA samples were prepared from liver tissue for toxicogenomic analysis. None of the profiles of the test substance, which is described in the literature as genotoxic carcinogen, was classified as expected, all profiles being classified as non-carcinogen or even unclassified. Thus, the prediction for the test substance was false negative and the method is not applicable for the test substance. (Ellinger-Ziegelbauer et al., Mutation Research 637, 23–39, 2008)

Human data

In a Swedish retrospective study, the state of health was investigated in three subgroups of power generator workers; 192 workers were exposed to 4,4'-MDA between 1963 and 1985, 237 workers were possibly exposed and 121 workers were unexposed. The concentration of 4,4'-MDA, the exposure route and time of exposure were not registered. No cancer cases occurred in the exposed group, two cancer cases occurred in the possibly exposed group, and four cases, included one case of bladder cancer, occurred in the unexposed group. The authors concluded that there was no statistically significant evidence of an increased overall or bladder cancer risk compared to the total population.

(Sélden A, et al., Int Arch Occup Environ Health 63, 403-8, 1992)

Ten workers exposed to 4,4'-MDA from 7 days to 2.5 months between 1967 and 1976 were followed. The concentration supposed to be inhaled ranged from 0.04-3.11 mg/m3. After developing acute jaundice the workers left the factory. Twenty-three years after intoxication, one worker was diagnosed with bladder cancer. Since the average latency period for aromatic amine-induced cancer has been suggested to be about 20 years, occurrence of bladder cancer has been observed in other persons occupationally exposed to 4,4'-MDA, and on the basis of animal data, the authors concluded that this finding added weight to the suggestion that 4,4'-MDA is carcinogenic in humans.

(Liss GM et al., Am J Ind Med 26, 117-24, 1994)

A follow-up investigation of 179 white male deaths among employees with potential exposure to epoxy resins and amine hardeners who had ever worked for more than one month in areas with potential exposure to 4,4'-MDA was conducted. Forty-six persons of this group died with malign neoplasms. The proportional mortality rate amongst these persons revealed a statistically significant excess of cancer of the large intestine, cancer of the bladder, lymphosarcoma and reticulosarcoma compared to the whole population. In a proportional cancer mortality ratio analysis the excess of bladder cancer remained significantly elevated. On the basis of these findings, the U.S. National Institute for Occupational Safety and Health (NIOSH) suggested an association between bladder cancer and work in areas with past or present potential exposure to 4,4'-MDA.

(NIOSH, HETA 82-146-1388, 1983)